The objectives of this proposal are to elucidate the molecular mechanisms of catalysis and regulation of dopamine-Beta-hydroxylase. Experimental approaches to the elucidation of catalytic mechanism will include (i) steady state kinetic studies employing deuterium and tritium labelled substrates to obtain an intrinsic isotope effect for the C-H bond cleavage step and to assign rate constants to individual steps in the overall mechanism; (ii) rapid quench studies to quantitate the stoichiometry of norepinephrine formation to enzyme subunit in a single enzyme turnover and to provide an independent assignment of rate constants for comparison with steady state investigations, (iii) analysis of structure-reactivity correlations and Alpha- and Beta-secondary isotope effects under experimental conditions leading to full isolation of the C-H cleavage step; (iv) investigation of alternate sources of oxygen (e.g. iodosobenzene) capable of supporting substrate hydroxylation; and (v) full characterization of the mechanism of dopamine-Beta-hydroxylase inhibition by a new class of substrates/inhibitors, Beta-substituted phenethylamines. Experimental approaches to the elucidation of regulation will include (i) an examination of effector induced changes in kinetic isotope effect for dopamine hydroxylation (ii) the fractionation and characterization of soluble components of bovine chromaffin vesicles in an effort to identify putative "in vivo" effectors; (iii) characterization of the molecular weight properties of soluble and membrane-bound enzyme to test the hypothesis that these enzyme forms may differ solely in their oligomeric structure and (iv) the measurement of rate constants and isotope effects for dopamine uptake and hydroxylation by intact chromaffin vesicles. Dopamine-Beta-hydroxylase plays a key role in the biosynthesis of the hormone/neurotransmitter norepinephrine. Enzyme is released into cerebronspinal fluid and blood plasma concomitant with sympathetic nervous system activity and a large number of clinical studies have been conducted with the goal of correlating levels of circulating enzyme with disease states such as schizophrenia and hypertension. The mechanistic studies described in this proposal should aid both in the interpretation of clinical data and the design of pharmacologically active compounds.